Identification of the DNA binding domain of the phage lambda cII transcriptional activator and the direct correlation of cII protein stability with its oligomeric forms.

Ho, Yen Sen; Mahoney, Michael; Wulff, Daniel L.; Rosenberg, Martin

doi:10.1101/gad.2.2.184

Cited by 35 publications

(18 citation statements)

References 45 publications

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“…Therefore, although nonspecific binding of the mutant proteins was not identical to that of the wild type, this binding was not abolished. These results are similar to those observed for mutations in the recognition helix of the cII protein, which eliminate sequence-specific binding but do not eliminate nonspecific binding (25).…”

Section: Vol 182 2000supporting

confidence: 78%

“…However, the transcriptional activity of the spoIIA and spoIIE promoters positively regulated by Spo0A was decreased by this substitution. This result is consistent with the S210 residue being part of the HTH DNA-binding domain, as many mutations within and around the HTH DNA-binding domain in other proteins are known to affect transcriptional activation without necessarily affecting binding (25,30).…”

Section: Vol 182 2000supporting

confidence: 76%

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Mutational Analysis of Conserved Residues in the Putative DNA-Binding Domain of the Response Regulator Spo0A of Bacillus subtilis

Hatt

Youngman

2000

J Bacteriol

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The Spo0A protein of Bacillus subtilis is a DNA-binding protein that is required for the expression of genes involved in the initiation of sporulation. Spo0A binds directly to and both activates and represses transcription from the promoters of several genes required during the onset of endospore formation. The C-terminal 113 residues are known to contain the DNA-binding activity of Spo0A. Previous studies identified a region of the Cterminal half of Spo0A that is highly conserved among species of endospore-forming Bacillus and Clostridium and which encodes a putative helix-turn-helix DNA-binding domain. To test the functional significance of this region and determine if this motif is involved in DNA binding, we changed three conserved residues, S210, E213, and R214, to Gly and/or Ala by site-directed mutagenesis. We then isolated and analyzed the five substitution-containing Spo0A proteins for DNA binding and sporulation-specific gene activation. The S210A Spo0A mutant exhibited no change from wild-type binding, although it was defective in spoIIA and spoIIE promoter activation. In contrast, both the E213G and E213A Spo0A variants showed decreased binding and completely abolished transcriptional activation of spoIIA and spoIIE, while the R214G and R214A variants completely abolished both DNA binding and transcriptional activation. These data suggest that these conserved residues are important for transcriptional activation and that the E213 residue is involved in DNA binding.The Spo0A protein of Bacillus subtilis is a member of the phosphorylation-activated response regulator family of twocomponent signal transduction proteins (3,16,35,42) and is required in a signal transduction pathway that controls the initiation of sporulation in response to nutrient limitation (10, 47). Spo0A functions as both a repressor and activator of gene transcription during the transition from exponential growth to the stationary phase (43) and during the early stages of sporulation (39,45,47). Phosphorylated Spo0A represses the transcription of a key transition state regulator, AbrB, by binding to the promoter of the abrB gene (43,45). In addition, phosphorylated Spo0A is required for the activation of transcription of key sporulation-specific genes, which include spoIIA (47), spoIIE (53), and spoIIG (6, 7). Spo0A has been shown to bind to specific sequences in the DNA upstream of the promoters it positively regulates and downstream of the promoters it negatively regulates (41). The Spo0A recognition sequence in DNA is referred to as the 0A box and consists of the 7-bp sequence 5Ј-TGTCGAA-3Ј (6, 43).The response regulator family of two-component signal transduction proteins is characterized by a conserved aminoterminal phosphoacceptor domain and unique carboxyl-terminal effector domains (3, 35, 42) (see Fig. 1A). Response regulators that function as transcription factors, i.e., OmpR (38), UhpA (28), and others (3, 35, 42), generally consist of two domains: the phosphoacceptor domain and an effector domain containing DNA-binding and...

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Section: Vol 182 2000supporting

confidence: 78%

Section: Vol 182 2000supporting

confidence: 76%

Mutational Analysis of Conserved Residues in the Putative DNA-Binding Domain of the Response Regulator Spo0A of Bacillus subtilis

Hatt

Youngman

2000

J Bacteriol

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“…These data, and the lack of homology to the Ecr70 -35 sequence, imply that C is involved in transcript initiation. Based on its DNA-binding activity (9), it is most likely that C acts as an accessory factor to polymerase, analogous to the lambda cI and clI proteins (34,35,36) or host CAP, OmpR, and other activator proteins (15,39,64), which bind in or upstream from the nonconsensus -35 regions of specific promoters, allowing their recognition and activation by host polymerase. Experiments in progress with extracts from a C-overproducing strain to initiate transcription from P1ys in vitro (W. Margolin, unpublished results) should help to define the precise biochemical role of C in promoter activation.…”

Section: Resultsmentioning

confidence: 99%

Bacteriophage Mu late promoters: four late transcripts initiate near a conserved sequence

1989

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Late transcription of bacteriophage Mu, which results in the expression of phage morphogenetic functions, is dependent on Mu C protein. Earlier experiments indicated that Mu late RNAs originate from four promoters, including the previously characterized mom promoter. Si nuclease protection experiments were used to map RNA 5' ends in the three new regions. Transcripts were initiated at these points only in the presence of C and were synthesized in a rightward direction on the Mu genome. Amber mutant marker rescue analysis of plasmid clones and limited DNA sequencing demonstrated that these new promoters are located between C and lys, upstream of I, and upstream of P within the N gene. A comparison of the promoter sequences upstream from the four RNA 5' ends yielded two conserved sequences: the first (tA . . cT, where capital and lowercase letters indicate 100 and 75% base conservation, respectively), at approximately -10, shares some similarity with the consensus Escherichia coli C70 -10 region, while the second (ccATAAc CcCPuG/Cac, where Pu indicates a purine), in the -35 region, bears no resemblance to the E. coli -35 consensus. We propose that these conserved Mu late promoter consensus sequences are important for C-dependent promoter activity. Plasmids containing transcription fusions of these late promoters to lacZ exhibited C-dependent lI-galactosidase synthesis in vivo, and C was the only Mu product needed for this transactivation. As expected, the late promoter-lacZ fusions were activated only at late times after induction of a Mu prophage. The C-dependent activation of lacZ fusions containing only a few bases of the 5' end of Mu late RNA and the presence of altered promoter sequences imply that C acts at the level of transcription initiation.Viruses have evolved a number of regulatory schemes to ensure efficient production of progeny particles during lytic development. A common strategy is to sequentially activate and inhibit transcription of different sets of viral genes. In general, genes for DNA replication are transcribed early, while those for'virion formation and release are expressed later. Activation of new sets of genes often involves utilization of new promoter sequences which are recognized by new virus-specific polymerases or by altered forms of the host polymerase. For example, Escherichia coli phages T7 and N4 synthesize their own RNA polyrmerases (12, 18), whereas Bacillus phage SPOl and E. coli phage T4 produce phage-encoded sigma factors which substitute for the host sigma subunit (14, 42). E. coli phage lambda uses two additional mechanisms: it produces accessory DNA-binding proteins, cI and cII, which enhance initiation by host RNA polymerase holoenzyme at specific promoters (55,69), and it modifies the host RNA polymerase complex with phage proteins N and Q to achieve antitermination and thus elongation of short constitutive leader transcripts (1,19

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“…Although our results indicate that HflD enhances degradation of CII by bringing it to the vicinity of membrane-bound FtsH, it might participate in lysogeny control also by sequestering CII from the target promoter (43). In this latter respect, it could be regarded as similar to RseA, a membrane-bound anti-sigma E factor (44).…”

Section: Discussionmentioning

confidence: 91%

Revisiting the Lysogenization Control of Bacteriophage λ

Kihara

Akiyama

Ito

2001

Journal of Biological Chemistry

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Upon infection to the Escherichia coli cell, the genome of bacteriophage either replicates to form new progenies (lytic growth) or integrates into the host chromosome (lysogenization). The CII protein is a key determinant in the lysis-lysogeny decision. It is a short-lived transcription activator for the genes essential for lysogeny establishment. In this study, we isolated a new class of hfl (high frequency lysogenization) mutants of E. coli, using a new selection for enhancement of CIIstimulated transcription. The gene affected was termed hflD, which encodes a protein of 213 amino acids. An hflD-disrupted mutant indeed showed an Hfl phenotype, indicating that HflD acts to down-regulate lysogenization. HflD is associated peripherally with the cytoplasmic membrane. Its interaction with CII was demonstrated in vitro using purified proteins as well as in vivo using the bacterial two-hybrid system. Pulsechase examinations demonstrated that the HflD function is required for the rapid in vivo degradation of CII, although it interfered with FtsH-mediated CII proteolysis in an in vitro reaction system using detergent-solubilized components. We suggest that HflD is a factor that sequesters CII from the target promoters and recruits it to the membrane where the FtsH protease is localized.

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Identification of the DNA binding domain of the phage lambda cII transcriptional activator and the direct correlation of cII protein stability with its oligomeric forms.

Cited by 35 publications

References 45 publications

Mutational Analysis of Conserved Residues in the Putative DNA-Binding Domain of the Response Regulator Spo0A of Bacillus subtilis

Mutational Analysis of Conserved Residues in the Putative DNA-Binding Domain of the Response Regulator Spo0A of Bacillus subtilis

Bacteriophage Mu late promoters: four late transcripts initiate near a conserved sequence

Revisiting the Lysogenization Control of Bacteriophage λ

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